PROJECT SUMMARY/ABSTRACT The American Cancer Society estimates that head and neck cancer will account for 61,760 new cancer cases and 13,190 cancer-related deaths in the United States in 2016, more than 90% of which will be squamous cell carcinoma (HNSCC). While cigarette smoking and alcohol consumption are primary known risk factors, human papillomavirus (HPV) is another major independent risk factor that is detectable in approximately 25% of cases. Importantly, HPV+ HNSCC is considered to be biologically distinct, and is generally associated with favorable outcomes. However, despite the comparative prognostic advantage, survival remains relatively poor among all patients with advanced stage disease, underscoring the urgent need for discovery, development and translation of novel biomarkers to facilitate early detection of HNSCC and bolster the chance for positive patient outcomes. Exosomes may offer a new avenue for discovery and development of novel HNSCC biomarkers. Exosomes are nano-sized (40-150 nm), membrane-encapsulated vesicles that are released by both normal and malignant cells into the extracellular space and function as intercellular signaling vectors through the horizontal transfer of biologic molecules. Importantly, they are detected in essentially all extracellular biofluids, including saliva. Indeed, mounting evidence indicates that cancer-derived exosomes enable a tumor to manipulate its microenvironment, potentially contributing to immune evasion, tumor growth, invasion, and metastatic spread, making them particularly attractive biomarker sources. Salivary exosomes have been theorized as a potentially powerful biomarker source for head and neck cancer. However, to date, there has been an extreme paucity of work conducted in this arena, which has focused solely on the exosomal proteome. We recently demonstrated the strong potential for exosomal miRNA cargo as biomarkers of HPV- negative HNSCC, in which we observed dramatic differences in exosomal miRNA cargo between HPV- negative HNSCC cells compared to that from primary oral epithelial cells from healthy donors, with a high degree of overlap in exosomal miRNA profiles between the 4 distinct HNSCC cell lines. However, it is unclear if the findings of the aforementioned can be generalizable to HPV+ HNSCC, given their discrete biology ? indeed, our preliminary salivary data hints that they may not. Thus, there is a need to fill this gap in our understanding of exosomal miRNA signaling in HNSCC by expanding our previous work to include HPV+ HNSCC cell lines, and translating a set of candidate HNSCC biomarkers, based on our previous findings and the proposed cell culture work detailed herein, into an expanded set of saliva samples, with an emphasis on early stage disease. We believe that this proposed work is poised to bolster our comprehension of the biology of HPV+ HNSCC and to elucidate novel biomarkers that can be harnessed for early detection and reduce the morbidity and mortality that stems from this devastating disease.